Ultracentrifugation is the reference method for separating lipoproteins from plasma, although other methods have also been reported, e.g., electrophoretic methods which depend on their differing surface charges and molecular sizes, precipitation methods where insoluble complexes are formed with polyanions and divalent cations, gel or membrane filtration methods which separate on the basis of molecular size, and isolation methods using antibodies to apolipoproteins. Three different ultracentrifugation operations are required to effect the separation:
(1) 16 hours at a plasma density of 1.006 grams per milliliter and 10,000.times.gravity, followed by recovery of VLDL by a technique called tube-slicing; PA1 heparin--Mn.sup.++ PA1 dextran sulfate 500--Mg.sup.++ PA1 sodium phosphotungstate--Mg.sup.++ PA1 heparin--Ca.sup.++ and PA1 concanavalin A--polyethylene glycol 6000.
(2) 20 hours at a plasma density of 1.063 grams per milliliter and 10,000.times.gravity followed by a density adjustment and recovery of HDL; and
(3) at a plasma density of 1.210 grams per milliliter to recover HDL.
The isolated lipoprotein fractions are then quantitatively recovered, reconstituted to the original volume of the plasma aliquot, and quantitated by measurement of their cholesterol, triglyceride, phospholipid or protein content (Textbook of Clinical Chemistry, Tietz, Saunders, 1986, page 876).
Various precipitation techniques are recommended for the determination of HDL-Cholesterol in small volumes of plasma although, again, ultracentrifugation is the reference method. The precipitation methods use divalent cations and sulfated polysaccharides or sodium phosphotungstate to precipitate all of the lipoproteins except HDL. Centrifugation can then be used to remove the precipitated lipoproteins, after which HDL remaining in the supernatant can be quantitated by its cholesterol content. Examples of precipitating reagents include:
(ibid., page 879)
The use of affinity column chromatography to isolate .alpha. and .beta. lipoprotein fractions from serum and plasma has been described (Clinical Chemistry, Volume 28, No. 7, pages 51 et seq., 1982; see, also, a system that has been offered under the trade designations LDL-Direct and LDL-Direct Plus). A polypropylene column equipped with porous polyethylene filter discs and packed with a heparin-agarose affinity medium was used to produce an .alpha.-lipoprotein fraction (which contains all the lipoproteins that do not specifically bind to the column--said to be essentially the non-atherogenic components, similar to the HDLs obtained by ultracentrifugation) and a .beta.-lipoprotein fraction. The .beta.-fraction, which is ultimately desorbed from the column with saline, is said to be essentially the LDL (low density lipoprotein) and VLDL (very low density lipoprotein) fractions obtained by ultracentrifugation. The .alpha.- and .beta.-fractions were then analyzed for cholesterol or the like and the .beta.-:.alpha.-lipoprotein cholesterol ratio was determined. This ratio, it is said, may better indicate a patient's risk of stroke or coronary heart disease than the value for HDL cholesterol alone.
It has also been reported ("Determination of high density lipoprotein cholesterol in venous and capillary whole blood", Lippi, U. et al., Journal of Lipid Research, Volume 29, 1988, pages 112 et seq.) that HDL Cholesterol can be determined in whole blood anticoagulated with EDTA, using a precipitation mixture containing 99.9 grams per liter of polyethylene glycol 6000, 37.4 milligrams per liter of dextran sulfate and 2.6 millimoles per liter of MgCl.sub.2. The procedure is described as involving separating HDL from plasma or whole blood by adding 25 microliters of plasma or 50 microliters of whole blood to 250 microliters of the precititation mixture, gentle mixing for a few seconds, incubating for 10 minutes and centrifuging at 1,500.times.gravity for 10 minutes and determination of HDL Cholesterol by adding 200 microliters of supernatant to 1.0 milliliter of the enzymatic cholesterol reagent and reading absorbances at 500 nanometers.